A secreted phospholipase A_(2) (BmsPLA_(2)) regulates melanization of immunity through BmDDC in the silkworm Bombyx mori

Insect immune-associated phospholipase A_(2) (PLA_(2)) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA_(2) on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA_(2) gene (BmsPLA_(2)), and its protein had characteristic features of secreted PLA_(2) (sPLA_(2)). After injection of bacteria, BmsPLA_(2) expression and sPLA_(2) activity in hemolymph significantly increased. BmsPLA_(2) fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA_(2) was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA_(2) by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA_(2) overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA_(2) inhibitor dosage. These results demonstrated that BmsPLA_(2) plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA_(2) after injection of bacteria, implying that BmsPLA_(2) was activated prior to BmDDC. Moreover, the alteration of BmsPLA_(2) by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA_(2) regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.